Corticotropin-releasing factor overproducing transgenic mice

ABSTRACT

In accordance with the present invention, there are provided CRF overproducing transgenic mice which exhibit endocrine abnormalities involving the hypothalamic-pituitary-adrenal axis, such as elevated plasma levels of ACTH and glucocorticoids. The transgenic mice of the present invention represent a genetic model of CRF overproduction, providing a valuable tool for investigating the long term effects of CRF excess and dysregulation in the central nervous system.

ACKNOWLEDGEMENT

This invention was made with Government support under Grant NumberDK26741, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

This application is a divisional of application Ser. No. 08/068,754,filed on May 28, 1993, issued as U.S. Pat. No. 6,023,011.

FIELD OF THE INVENTION

The present invention relates to the generation of transgenic mice anduses therefor.

BACKGROUND OF THE INVENTION

Corticotropin-releasing factor (CRF) is a 41-residue hypothalamicpeptide which stimulates the secretion and biosynthesis of pituitaryACTH leading to increased adrenal glucocorticoid production. CRF wasoriginally isolated and characterized on the basis of its role in thishypothalamic-pituitary-adrenal axis (HPA) [Vale et al., Science Vol.213:1394-1397 (1981)]. More recently, however, it has been found to bedistributed broadly within the central nervous system (CNS) as well asin extra-neural tissues such as the adrenal glands and testes [Swansonet al., Neuroendocrinology Vol. 36:165-186 (1983); Suda et al., J. Clin.Endocrinol. Metab. Vol. 58:919-924 (1984; Fabbri and Dufau,Endocrinology Vol. 127:1541-1543 (1990)], where it may also act as aparacrine regulator or neurotransmitter.

In addition to its critical role of mediating HPA axis activation, CRFhas been shown to modulate behavioral changes that occur during stressresponse. Many of these behavioral changes have been shown to occurindependently of HPA activation in that they are insensitive todexamethasone treatment and hypophysectomy [Britton et al., Life Sci.Vol. 38:211-216 (1986); Britton et al., Life Sci. Vol. 39:1281-1286(1986); Berridge and Dunn, Pharm. Bioch. Behav. Vol. 34:517-519 (1989)].In addition, direct infusion of CRF into the CNS mimics autonomic andbehavioral responses to a variety of stressors [Sutton et al., NatureVol. 297:331-333 (1982); Brown and Fisher, Brain Res. Vol. 280:75-79(1983); Stephens et al., Peptides Vol. 9:1067-1070 (1988); Butler etal., J. Neurosci. Vol. 10:176-183 (1990)]. Furthermore, peripheraladministration of CRF or the CRF antagonist, α-helical CRF 9-41, failedto effect these changes, thus supporting a central role for CRF in suchfunctions.

Central administration of CRF in rodent animal models produces effectsthat correlate with a state of anxiety such as a reduction inwillingness to investigate unfamiliar surroundings [Sutton et al.,supra; Sherman and Kalin, Pharm. Biochem. Behav. Vol. 26:699-703 (1987);Berridge and Dunn, supra; Butler et al., supra], decreased sleep[Sherman and Kalin, supra], enhanced fear responses [Sutton et al.,supra; Butler et al., supra), decreased food consumption [Morely andLevine, Life Sci. Vol. 31:1459-1464 (1982)] and suppressed sexualbehavior (Sirinathsinghji et al., Nature Vol. 305:232-235 (1983)). Thesechanges are similar to behavioral changes observed upon exposure toacute and chronic stressors, and resemble changes that occur in humanaffective disorders such as the symptom complex characteristic of majordepressive disorder, panic disorder and anorexia nervosa [Kaye et al.,J. Clin. Endocrinol. Metab. Vol. 64:203-208 (1987); Gold et al., N.Engl. J. Med. Vol. 319:413-420 (1988); Kathol et al., Psych. Clin. N.Amer. Vol. 22:335-348 (1988); Nemeroff, C. B., Pharmacpsychiat Vol.21:76-82 (1988)].

Currently, a great deal of interest in CRF has been generated inconnection with the pathophysiology of mental illness. For example, CRFhypersecretion has been linked to some individuals diagnosed with majordepression [Nemeroff et al., Science Vol. 226:1342-1344 (1984)]. Whileall studies do not support the suggestion that cerebrospinal fluid CRFlevels are altered in this group of individuals, most researchers agreethat HPA axis responsivity in these individuals is abnormal. Indeed, alarge portion of individuals diagnosed with depression have elevatedcortisol levels [Roy-Byrne et al., Am. J. Psychiatry Vol. 143:896-899(1987); Kling et al., J. Clin. Endocrinol. and Metab. Vol. 72:260-271(1991)). Moreover, in major depression [Holsboer et al., N. Engl. J.Med. Vol. 311:1127 (1984)] and panic disorder (Roy-Byrne, supra] CRFadministration results in a blunted ACTH response, suggesting that thepituitary is properly restrained, presumably by the negative feedbackeffect of elevated levels of glucocorticoids. In view of these findings,it has been suggested that the hypercortisolism in major depression isdue to abnormal CRF secretion within the CNS [Gold et al., Psychiat.Clinics of N. Amer. Vol. 11:327-335 (1988)].

In order to more fully investigate the role of abnormal CRF secretionwithin the CNS, it would be desirable to have available animal modelswith which to study the effects of CRF hypersecretion.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have developed a transgenicmouse model exhibiting chronic CRF hypersecretion. These animals expresshigh levels of ACTH and corticosterone throughout their life span anddevelop a Cushing's syndrome phenotype due to excess glucocorticoidproduction.

Although transgenic animals show normal plasma levels of CRF, numerouscentral nervous system sites show elevated CRF gene expression. The factthat these animals show chronic hypersecretion of CRF and thus,hyperactivation of the pituitary-adrenal axis, makes them a good modelto investigate the role of CRF in long-term behavioral changes,particularly with respect to anxiogenic effects.

As described in greater detail herein, the hypothesis that persistentcentral CRF hypersecretion produces behavior characteristic of anxietywas tested. Measurements of anxiety were made using an ElevatedPlus-Maze, a test that is based on the natural aversion of rodents foropen spaces [Pellow et al., J. Neurosci. Methods Vol. 14:149-167 (1985);Lister, R. G., Psychopharmacology Vol. 92:180-185 (1987)]. In addition,the effect of exposure to an unfamiliar (novel) environment on locomotoractivity was assessed. The effect of social aggression on theperformance of CRF transgenics was also examined in order to determinethe reactivity of this animal model to a psychological stressor.Finally, to test whether brain CRF plays a role in mediating behaviorcharacteristic of anxiety among CRF transgenics, the stress protectiveactions of the centrally administered CRF antagonist, α-helical CRF9-41, was examined.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B present the mean (±SEM) locomotor activity (FIG. 1A) andcrossover frequency (FIG. 1B) of control (□; n=10) and transgenic CRF(; n=12) mice placed individually for 30 minutes in novel photocellcages. * p<0.05; ** p<0.01.

FIG. 2 presents the mean (±SEM) locomotor activity of untreated control(∘; n=10) or CRF transgenic (□; n=12) mice and of pre-stressed control(; n=6) or CRF transgenic (▪; n=7) mice placed individually for 30minutes in novel photocell cages. * p<0.05 vs. untreated CRF transgenicgroup; ** p<0.0l vs. prestressed CRF transgenic mice over a 5 minutetest on the elevated plus-maze.

FIGS. 3A and 3B present the mean (±SEM) percent of time spent on theopen arms (FIG. 3A) and overall activity (FIG. 3B) of control (n=8) orCRF transgenic (n=10) mice over a 5 minute test on the elevatedplus-maze. * p<0.05.

FIGS. 4A and 4B present the mean (±SEM) percent of time spent on theopen arms (FIG. 4A) and overall activity (FIG. 4B) during a 5 minutetest on the elevated plus-maze following pre-treatment with CRFantagonist of both control (0 μg pre-treatment, n=7; 1 μg, n=5; 5 μg,n=5) and CRF transgenic (0 μg pre-treatment, n=7; 1 μg, n=5; 5 μg, n=6)mice. * p<0.05 vs. vehicle-treated control groups; +p<0.05 vs.vehicle-treated CRF transgenic group.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided transgenicmice containing exogenous DNA encoding corticotropin releasing factor(CRF). Exogenous DNA employed in the practice of the present inventioncomprises the rat CRF gene under the expression control of either aninducible promoter (e.g., the mouse metallothionein (MT) promoter), or aconstitutive promoter.

CRF genes employed in the practice of the present invention may beobtained by isolating them from genomic sources, by preparation of cDNAsfrom isolated mRNA templates, by direct synthesis, or by somecombination thereof. The published sequences of numerous CRF genesgreatly facilitates obtaining a gene encoding CRF, and this invention isnot limited to the use of any particular gene. However, the rat CRF geneis presently preferred.

To be expressed, the structural gene must be coupled to a promoter in afunctional manner, i.e., operably associated therewith. If aconstitutive promoter is used, a viral promoter, such as the SV40 earlypromoter, is preferred. The MT promoter, while often referred to as an"inducible" promoter, can alternatively be described as"semi-constitutive" since it is "on" all of the time, even though itsactivity is boosted by heavy metal ions. Such promoters, as well as pureinducible promoters, may also be used. Promoter/regulatory sequences maybe used to increase, decrease, regulate or target expression to certaintissues or to certain stages of development. The promoter employed neednot be a naturally occurring promoter.

The CRF gene may be introduced by zygote microinjection, as describedbelow. Alternatively, the CRF gene may be introduced into non-germlinecells (somatic cells) in a fetal, new born, juvenile or adult animal byretroviral vector mediated genetic transformation of somatic cells ofone or more tissues within the animal.

The widespread distribution of CRF in brains of normal animals has leadto a heightened interest in the role CRF plays in regulating andintegrating complex behavior [Swanson et al., supra; Imaki et al., BrainRes. Vol. 496:35-44 (1989); Imaki et al., Brain Res. Vol. 547:28-36(1991)]. The relative importance of CRF located in distinct brainregions is unclear, although CRF injection into specific sites, such asthe locus coeruleus (Butler et al., supra) and amygdala [Weiss et al.,Brain Res. Vol. 372:345-351 (1986); Liang and Lee, PsychopharmacologyVol. 96:232-236 (1988)] have been implicated in effecting distinctbehavioral responses. CRF expression in the transgenic model describedherein is clearly elevated in a number of sites in the brain, althoughperipheral plasma levels of CRF do not appear to be elevated. Thetransgenic mice of the invention display markedly elevated levels of CRFmRNA in nearly all areas where expression is shared in common withcontrols. In addition, CRF transgenic mice of the invention exhibitprominent levels of mRNA expression in some regions believed to be sitesof CRF expression in the rat, whereas CRF was not detectable in suchregions in control mice. Regions where mRNA expression was observed inthe transgenic mice includes the supraoptic and dorsomedial nuclei ofthe hypothalamus, lateral hypothalamic area, substantia innominata,vestibular complex, and the lateral reticular nucleus. Moreover, anumber of regions not previously identified as sites of CRF expressioncontain robust mRNA levels in the transgenic mice. Regions where suchmRNA expression was observed include the arcuate nucleus of thehypothalamus, the subfornical organ, lateral habenula, the granule celllayer of the dentate gyrus, the dorsal subiculum, and the deep nuclei ofthe cerebellum.

Daily, repeated administration of CRF has been employed as a means ofmodeling the chronic state of CRF activation reported to accompanyaffective and appetitive psychopathology in human clinical populations[see, for example, Hotta et al., Life Sci. Vol. 48:1483-1491 (1991)]. Byfollowing this course for several days (or weeks), one can observediminished weight gain, hypogonadism and persistenthypothalamic-pituitary-adrenal axis activation, which resembles thepattern of psychiatric symptomatology observed in depression or anorexianervosa. This relation is supported by the present data in which animalswith an intrinsic overproduction of CRF exhibit unprovoked hyperactivityto environmental stressors. Furthermore, while these animals have aCushing's-like phenotype associated with glucocorticoid excess, thepresent state of anxiety can be attributed to neurotropic actions of CRFwithin the brain since the enhanced emotionality of the transgenic micewas reversed with a centrally administered CRF receptor antagonist.Hence, this new animal model is well suited for testing neurogenichypotheses in the etiology of human affective disorders.

Because pituitary ACTH and hypothalamic paraventricular CRF expressionis suppressed by glucocorticoids, [Jingami et al, Endocrinology Vol.117:1314-1320 (1985)] it was unclear whether long term overproduction ofCRF by transgenic mice would lead to the development of ACTH-dependentCushing's syndrome in these animals. In addition, regulatory mechanisms,such as desensitization of CRF receptors, might also modulate theeffects of excess CRF production. However, as described herein, atransgenic animal model exhibiting chronic CRF expression has beendeveloped, leading to the development of a Cushing-like syndrome inmice. These animals show elevated CRF expression due to the presence ofa rat CRF transgene, which results in chronic pituitary stimulation ofACTH release and elevated glucocorticoid levels. In addition, animalsthat express the transgene display physical features found amongpatients with Cushing's syndrome, such as obesity, hair loss, thin skin,muscle wasting, and reduced fertility. Several of these features appearto be due in part to increased circulating corticosterone levels, sinceadrenalectomized transgenic animals revert to a normal phenotype withrespect to hair and skin changes within a month after adrenalectomy.

Treatment with zinc to induce higher expression of the CRF transgene didnot change basal corticosterone levels, which may indicate that CRFstimulation of ACTH and, consequently, corticosterone was maximal inthis setting or that the regions of expression of the CRF transgene wereinsensitive to metal induction. Since transgene expression was notcompared in the absence and presence of metal induction, it is not knownwhether the MT-CRF construct in this line is sensitive to suchtreatments. However, it appears that treatment with zinc did not resultin detectable expression of the transgene in several regions where itwas expected to occur, such as liver and kidney. Although peripheralplasma CRF levels were not detectably elevated in the transgenics, CRFgene expression in several brain regions was increased, which presumablyleads to increased ACTH and corticosterone levels in these animals.

Excess production of CRF has been implicated in the development ofcorticotrope hyperplasia and microadenoma formation [Cary et al., N.Engl. J. Med. Vol. 311:13-20 (1984); Kreiger, D. T., Endocr. Rev. Vol.4:22-43 (1983); Gertz et al., Endocrinology Vol. 120:381-388 (1987)].CRF transgenics were not found to have increased numbers ofACTH-staining cells in the pituitary, nor was a difference in pituitaryweight noted among the transgenics. It is possible that changes incorticotrope number may only become apparent in much older transgenicanimals or upon removal of glucocorticoid feedback.

CRF expression in transgenic mice was detected in all known sites ofendogenous CRF expression. In most of the regions, the level of CRF iselevated in the transgenics compared with that in control animals.Although no CRF was detected in the principal sites of normal MT geneexpression (liver and kidney), CRF was found in seminiferous tubules,where MT expression is under unique cadmium-insensitive regulation[Swapan et al., Mol. Endocrinol. Vol. 5:628-636 (1991)]. Thus, MT-CRFgene expression may be influenced by the MT promoter in the seminiferoustubules. CRF expression may occur normally in the pituitary, either atlow levels or under specific conditions, and the presence of thetransgene amplifies that potential to a level high enough to allowdetection. The fact that CRF has been detected in normal rat pituitariesby Northern blot analysis is consistent with this possibility [Thompsonet al., Mol. Endocrinol. Vol. 1:363-370 (1987)].

Overall, the majority of sites of CRF transgene expression are normalsites of endogenous expression, which suggests that gene elementscarried within the CRF-coding region or intron may be directingtissue-specific expression of the CRF hybrid transgene. Very fewtransgenes that employ the MT promoter fail to express in the liver[Kelley et al., Mol. Cell Biol. Vol. 8:1821-825 (1988)], although novelcell distribution patterns have been reported that appear to result fromthe combined influence of both genetic components of the fusion gene[Swanson et al., Nature Vol. 317:363-366 (1985)]; Russo et al., NeuronVol. 1:311-320 (1988)]. In particular, ectopic neuronal expression hasbeen reported from chimeric transgenes employing the MT promoter;however, the pattern of expression did not appear to be a uniqueproperty of the MT promoter alone, since not all MT fusion genes wereexpressed in neuronal sites [Russo et al. supra]. It is conceivable thatnovel sites of CRF expression in the transgenics may representenhancement of expression normally below the level of detection.

The sparse expression of the transgene in peripheral sites could explainthe observation that circulating CRF levels are not elevated in thetransgenics. Most brain regions considered to be normal sites of CRFexpression showed increased CRF hybridization in transgenic brains. Onenotable exception to this is the strength of the hybridization signal inthe paraventricular nucleus of the hypothalamus using a riboprobe thatdetects both endogenous CRF and the transgene. This may reflect the factthat endogenous CRF expression in the paraventricular nucleus exhibitsmarked glucocorticoid down-regulation, while extrahypothalamic areas,such as olfactory bulb, midbrain cerebral cortex, and brain stem, havebeen found to be relatively insensitive to perturbations incorticosteroid titers [Imaki et al., J. Neurosci. Vol. 11:585-599(1991)].

While there is limited expression in peripheral sites, the transgene isexpressed in brain regions that could provide CRF stimulation toACTH-producing cells in the pituitary, and thereby influence thedevelopment of the Cushing's phenotype. In addition to theCRF-containing cells located in the pituitary itself, theparaventricular nucleus, the arcuate nucleus, and the supraoptic nucleiin the hypothalamus are each capable of contributing to the CRF contentof the hypophyseal-portal vasculature, which could provide chronic CRFstimulation of pituitary ACTH secretion in the transgenic animals. Thus,increased levels of CRF mRNA at one or more sites in the hypothalamusand pituitary in these animals may be responsible for the elevated ACTHand glucocorticoid levels detected in plasma. Moreover, the finding thatCRF is expressed in the pituitary provides a plausible route ofparacrine or autocrine regulation of ACTH production by corticotropes.In addition, recent evidence obtained from normal animals indicates thatthe adrenal gland may modulate glucocorticoids via local ACTH productionfrom adrenal sources [Jones and Edwards, J. Physiol. Vol. 430:25-36(1990); Andreis et al., Endocrinology Vol. 128:1198-1200 (1991)]. Thefact that CRF transgene expression occurs in the adrenal gland in thetransgenics may provide an additional mechanism for paracrine regulationof glucocorticoid production that would not necessarily lead toelevations in plasma CRF, but could result in increased local CRF levelsand thereby effect ACTH and corticosterone production.

The transgenic mice described herein provide an opportunity toinvestigate the physiological consequences of overproduction of acentral neuropeptide, with numerous effects that modulate behavioral,autonomic, and neuroendocrine functions. The animals display a tissuedistribution of CRF expression that should yield exaggerated autocrineand paracrine stimulation. In addition to serving as a novel model ofCushing's disease, CRF transgenic mice may provide useful animal modelsof clinical depression, anorexia nervosa, and susceptibility to immunedysfunction; all syndromes postulated to involve alterations in centralor peripheral CRF systems [Gold et al., N. Engl. J. Med. Vol.319:413-420 (1988); Kaye et al., J. Clin. Endocrinol. Metab. Vol.64:203-208 (1987); Sternberg et al., Proc. Natl. Acad. Sci. USA Vol.86:4771-4775 (1989)].

In accordance with another embodiment of the present invention, there isprovided a method for treating a subject suffering from anxiety, saidmethod comprising modulating the expression and/or activity of CRF insaid subject. Such modulation can be accomplished in a variety of ways,e.g., the activity of CRF can be modulated by administering an effectiveamount of a CRF antagonist to the subject; or the expression of CRF canbe modulated by administering an effective amount of CRF antisense RNAto the subject, and the like.

For the above-contemplated administration, the modulating compounds canbe incorporated into a pharmaceutically acceptable formulation foradministration. Those of skill in the art can readily determine suitabledosage levels to be used.

As employed herein, the phrase "suitable dosage levels" refers to levelsof treating compound sufficient to provide circulating concentrationshigh enough to alter CRF expression and/or activity. Such aconcentration typically falls in the range of about 10 nM up to 2 μM;with concentrations in the range of about 100 nM up to 200 nM beingpresently preferred.

In accordance with a particular embodiment of the present invention,compositions to be administered are incorporated into a pharmaceuticallyacceptable carrier. Exemplary pharmaceutically acceptable carriersinclude carriers suitable for oral, intravenous, subcutaneous,intramuscular, intracutaneous, and the like administration.Administration in the form of creams, lotions, tablets, dispersiblepowders, granules, syrups, elixirs, sterile aqueous or non-aqueoussolutions, suspensions or emulsions, and the like, is contemplated.

In accordance with yet another embodiment of the present invention,there is provided a method of screening for compounds useful in thetreatment of Cushing's syndrome, said method comprising administeringtest compound(s) to a transgenic mouse of the invention, and monitoringfor improvement in symptoms characteristic of Cushing's syndrome.

In accordance with yet another embodiment of the present invention,there is provided a method of screening for compounds useful in thetreatment of anxiety, said method comprising administering testcompound(s) to a transgenic mouse of the invention and monitoring forimprovement in symptoms characteristic of anxiety.

The transgenic animals of the invention can also be used as a source ofcells for cell culture. Cells from the animals may advantageouslyexhibit desirable properties of both normal and transformed culturedcells; i.e., they will be normal or nearly normal morphologically andphysiologically, but can, like cells such as NIH3T3 cells, be culturedfor long, and perhaps indefinite, periods of time. Further, where thepromoter sequence controlling transcription of the recombinant genesequence is inducible, cell growth rate and other culturecharacteristics can be controlled by adding or eliminating the inducingfactor.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

Materials and Methods

Animals

CRF transgenic mice were generated by microinjection of ametallothionein-CRF (MT-CRF) gene construct, prepared as follows. Therat genomic CRF gene [see Thompson et al., Molec. Endocrinol. Vol.1:363-370 (1987)] extending from the Asp718 site in the 5'-untranslatedregion to the EcoRI site in the 3'-untranslated region (1.7 kilobasepair (kbp) fragment) was used in construction of the fusion gene. Therat CRF gene was digested with the restriction enzymes EcoRI and Asp718,followed by a fill-in reaction with Klenow fragment. The blunt endfragment was ligated into the SmaI site of a plasmid containing the 5'regulatory region of the mouse metallothionein-1 (MT-1) gene (1.8 kbp)(Palmiter et al., Science Vol. 222:809-814 (1983)) and the 3'untranslated region of the human growth hormone gene (0.65 kbp), whichcontains a polyadenylation signal sequence [DeNoto et al., Nucl. AcidsRes. Vol. 9:3719-3730 (1981)]. The MT-CRF gene was prepared formicroinjection by isolating a 4.1-kbp EcoRI fragment containing theMT-CRF fusion gene, which was purified by sucrose gradientcentrifugation. The fragment was microinjected into the male pronucleusof fertilized eggs (B6/SJL), and the injected eggs were transplanted topseudopregnant foster mothers following standard procedures (see, forexample, [Brinster et al., Proc. Natl. Acad. Sci. USA Vol. 82:4438-4442(1985)]. To identify transgenic founder animals, tail DNA from offspringwas screened by standard tail dot blot analyses using a ³² p randomlylabeled 0.65-kbp human GH 3'-fragment as a probe. Offspring of foundermice were screened using the polymerase chain reaction (PCR) andtransgene-specific primers to amplify tail DNA. Primers complementary tothe CRF transgene carboxyl region:

    5'-ACAGGAAACTGATGGAGATTATC-3';                             SEQ ID NO: 1

and the human GH gene:

    5'-TGGTGGGCACTGGAGTGGCAACT-3';                             SEQ ID NO: 2

were employed. PCR reactions were performed in 100-μl volumes in 50 mMKCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl, and 2.5 U Taq polymerase(Cetus Corp., Emeryville, Calif.). The following PCR conditions wereused: 1 cycle at 95° C. (5 min), 35 cycles at 95° C. (1 min), 55° C. (2min), and 72° C. (2 min), terminating with 1 cycle at 72° C. for 10 min.

A single transgenic founder male was used as the source of thistransgenic line and thus all animals are descendant offspring. Adultmale mice (transgenic and non-transgenic littermate controls) aged 2-9months and weighing 25-30 gm were housed singly in a pathogen-freetransgenic facility. Mice were given rodent chow and water ad libitumand kept on a 12 hour light/dark schedule with lights on from 0600 to1800.

Blood collection and hormone analyses

To obtain baseline hormone levels, plasma was obtained by retroorbitaleye bleeding from animals that were housed individually in coveredcages. Blood was collected at 0700 h within 45 sec of initialdisturbance of the cage, and samples were immediately placed on ice intotubes containing EDTA. Plasma was stored at -20° C. until assayed.Corticosterone levels were measured by RIA, using a rat/mouse [¹²⁵ I]corticosterone RIA kit (ICN Biomedicals, Costa Mesa, Calif.). Controlanimals were nontransgenic littermates or nontransgenic age- andsex-matched animals. Founder animals were bled before zinc treatment (25mM ZnSO₄ in the drinking water) and again 1 month later to determinewhether corticosterone levels were sensitive in induction of thetransgene by metal.

Basal ACTH levels were measured using a human ACTH two-siteimmunoradiometric assay (Nichols Institute, San Juan Capistrano, Calif.)with rat ACTH (1-39) as the standard.

Experiments involving animals were performed in accordance with the NIHguidelines for care and use of laboratory animals.

RNA analyses

Tissue was obtained from mice treated with ZnSo₄ (5 mg/kg, sc) 18 hbefore collection and frozen at -70° C. until use. Total RNA wasisolated by guanidinium isothyocyanate-cesium chloride centrifugation,as described by [Chrigwin et al., Biochemistry Vol. 18:5294-5299(1979)]. Total RNA (20 μg) was electrophoresed in 1.2% agarose-2.2 Mformaldehyde gels, blotted, and hybridized with a ³² P-labeled CRFantisense riboprobe, as described by [Imaki et al., supra). For PCRanalyses, total RNA from transgenic animals and control littermates wastreated with DNAse (1 U/μg) twice at 37° C. for 15 min before reversetranscription. DNAse was removed by treating the samples withproteinase-K (50 μg in 0.1% sodium dodecyl sulfate) for 15 min at 37°C., followed by phenol-chloroform extraction and ethanol precipitationof the RNA. RNA (3 μg) was reverse transcribed at 37° C. for 1 h usingpoly(dt) for cDNA priming and reverse transcriptase (avianmyeloblastosis virus; 4 U/μg; Life Sciences, St. Petersburg, Fla.). Tocontrol for potential false positive amplification of contaminatinggenomic DNA, duplicate RNA samples without reverse transcriptase wereincubated in parallel with the cDNA reactions, followed by PCRamplification. cDNA reactions (0.1 vol cDNA reactions) were heated to95° C. twice and subjected to PCR using the same primers and conditionsas those described for tail DNA amplification. PCR cycles were asfollows: 94° C. for 5 min, 40 cycles of 94° C. (1 min), 55° C. (2 min),and 72° C. (3 min), and a final elongation at 72° C. for 10 min. PCRproducts were electrophoresed, blotted, and probed with a 0.76 kbp BamHIfragment of the rat CRF gene, which includes the amplified region.

In situ hybridization and immunohistochemistry

Mice were anesthetized and perfused with saline, followed by 4%paraformaldehyde in 0.1 M borate buffer. Brains and peripheral tissueswere stored overnight at 4° C. in fixative containing 10% sucrose.Frozen sections (30 μm thick) were stored in antifreeze solution (30%polyethylene glycol, 20% glycerol, and 50% NaPO₄ (0.05 M)] until use.Tissue sections were mounted onto gelatin/poly-L-lysine-coated slides,and hybridizations were carried out using conditions previouslydescribed by [Imaki et al., supra]. [³⁵ S]CRNA probes were synthesizedusing SP6 (antisense) and T7 (sense) primed synthesis from a prepro-CRFcDNA plasmid template. Oligonucleotide hybridizations were performedusing a 42-mer complementary to the human GH fragment of the transgene:

    5'-TTA-GGA-CAA-GGC-TGG-TGG-GCA-CTG-GAG-TGG-CAA-CTT-CCA-GGG-3';(SEQ ID NO: 3)

Labeling and hybridization conditions using oligonucleotides were asfollows. Oligonucleotides were 3'-end labeled using terminaldeoxynucleotide transferase and [₃₅ S]deoxy-ATP to a specific activityof 3×10⁸ cpm/μg. Tissue sections were hybridized with 2.4×10⁶ cpm at 42°C. in 50% formamide, 5× Denhardt's, 4× SSC (1× SSC=0.15 M NaCl, 0.015 Msodium citrate), 10% dextran sulfate, 20 mM phosphate, 60 mMdithiothreitol, 125 μg/ml salmon sperm DNA, and 140 μg/ml transfer RNA.Slides were washed at 55° C. in 1× SSC for 45 min, dipped in NTB3nuclear track emulsion (1:1 with water; Eastman Kodak, Rochester, N.Y.),exposed for 8 days to 4 weeks, and developed. The sections werecounterstained with thionin.

Pituitary sections (20 μm) from a transgenic 15 mouse were incubatedwith rabbit αCRF-(1-21) (code 207-195) at 1:375 for 48 h at 4° C.,labeled with biotinylated donkey α-rabbit complexed withstreptavidin-Texas red followed by sheep αACTH at 1:1000 for 1 h at roomtemperature, and labeled with donkey α-sheep fluorescein isothiocyanate.To determine if the CRF positive calls colocalized with gonadotropes,sections were incubated simultaneously with rabbit α-CRF-(1-21) andeither mouse αLH at 1:500 or mouse αFSH at 1:1000 for 48 h at 4° C., andlabeled with goat α-rabbit lissamine-conjugated rhodamine and goatα-mouse 25 immunoglobulin G fluorescein isothiocyanate, respectively,for 1 h at room temperature.

Surgery and intracerebroventricular (ICV) microinjections

Mice were anesthetized with ketamine/xylazine (50 mg/kg, sc.) andmounted in a stereotaxic instrument with the incisor bar at -2.0 mm.Mice were implanted with a single cannula placed in the right lateralventricle. Guide cannulae (26 gauge, Plastics One, Roanoke, Va.) werepositioned 1.0 mm above the lateral ventricle (A/P at the bregma; D/V1.4 mm below the surface of the skull and M/L 1.1 mm lateral). Thecannulae were fixed to the skull using three 1.6 mm stainless steelscrews and dental cement. Animals were allowed to recover from surgeryfor a minimum of 5 days before testing, during which time 33 gauge dummycannulae were left inside the guide cannula. Intracerebroventricularinfusions were performed using 33 gauge infusion cannulae cut to extend1.0 mm beyond the end of the guide cannula. Dummy cannulae were removedand replaced by the infusion cannulae which were fitted to PE-50 tubingand connected to a 50 μl syringe. The infusion samples were delivered ina 2.0 μl volume over 30 sec. using an automated infusion pump. Infusioncannula were left in place for an additional 30 sec. to prevent effluxof infusion material and then replaced by the dummy cannulae for theduration of the experiment. To verify canulae placements, the brainswere removed, fixed in 10% formalin/10% sucrose and frozen just prior totissue sectioning on a freezing microtome.

Behavioral tests

All behavioral tests were performed between 1900 and 2400 hours duringthe active period of these animals.

Elevated Plus Maze

A four arm radial maze consisting of two opposing enclosed arms (30 cmhigh×30 cm long×5 cm wide) and two opposing exposed arms (30 cm×5 cm)was elevated on a pedestal 30 cm above the surface of a table andsituated in the center of a dimly lit room. Computer interfaced,infrared photocell beams situated around the perimeter and diagonallyacross the center of the maze monitored the amount of time spent in eachcompartment and provided a gross measure of overall activity. Mice wereplaced in the center of the maze facing an enclosed arm to begin thefive minute test period and the apparatus was cleaned with wetted towelsafter each test. Tests involving brain cannula infusions were performed5 minutes following the infusion of the test peptide or vehicle. The CRFantagonist, α-helical CRF 9-41 (available from American Peptide Inc.,Sunnyvale, Calif.), was dissolved in acidified water (pH 6.7) andanimals were infused with 1 or 5 μg of α-helical CRF 9-41 in a 2 μlvolume or given vehicle alone (2 μl).

Novel environment

A plexiglas box (33 cm long×23 cm wide×20 cm high) was equipped withcomputer interfaced, infrared photocell beams which trisected the lengthof the chamber. Mice which had not previously experienced thistesting-environment were allowed to explore the box for 30 minutesduring which time horizonal locomotor activity as well as movement fromone end of the box to the other was recorded. The boxes were cleanedwith water following each use. Experiments designed to test the effectof social defeat stress were performed 3-5 minutes following exposure ofthe test animal to the stressor. The social defeat stress consisted of abrief encounter between a test male (intruder) and a resident male(resident) which has been housed with a family comprised of a female andpups. The resident-intruder interaction took place in the resident malecage and in all cases, the intruder was placed in the cage for <1minute. At the first sign of aggressive behavior between the twoanimals, the intruder male was removed from the resident cage and housedsingly for 3-5 minutes before placement into the novel environmentchamber to measure locomotor activity.

Statistical Analysis

The overall two and three factor diagrams, as well as simple maineffects, were statistically analyzed by ANOVA. Individual means (±SEM)were compared using Student's t-tests.

EXAMPLE 1 Generation of MT-CRF Transgenic Mice

To test whether CRF overproduction leads to chronic activation ofpituitary ACTH production and excess glucocorticoid secretion,transgenic mice that overexpress CRF were developed. To avoid feedbackregulation of CRF transgene expression, the murine MT-1 gene promoterwas used in place of the natural CRF promoter [Durnam and Palmiter, J.Biol. Chem. Vol. 256:5712-5716 (1981)]. Because the polyadenylationsignal sequence used in CRF transcription was not included in thisclone, the 3'-untranslated region of the human GH gene, which contains apolyadenylation signal sequence, was included at the 3'-end of the CRFstructural gene.

From 100 pups, 11 transgenic animals were identified by tail DNA dotblot analysis. Founder animals were expected to express abnormally highsteady state levels of circulating CRF, leading to elevated ACTH releaseand increased adrenal corticosterone production. Elevated plasmacorticosterone levels were found in six founder animals, suggesting thatthe CRF transgene was expressed in these animals, leading to adrenaloverproduction of corticosterone. These founders displayed truncalobesity, with large adipose deposits, muscle wasting, bilateralsymmetric hair loss, and abnormally transparent skin. The severity ofthese Cushingoid features and the degree of endocrine dysfunction variedwidely among founders, without apparent correlation with transgene copynumber. Treatment of the founder animals with zinc (25 mM ZnSO₄ in thedrinking water) did not further raise the corticosterone levels in theseanimals.

Animals with increased corticosterone levels were selected for thegeneration of offspring. Animals in this line show physical changes thatcan be attributed to excess circulating corticosterone, such as hairloss, marked fat deposition, and thin skin [see Nelson in Endocrinology,DeGroot, ed., Saunders, Phila, Vol. 2:1660-1675 (1989)]. Transgeniclymphoid organs were markedly reduced in size, with a 3-fold differencenoted in spleen weight and an approximately 2-fold difference in thymusweight. Increased adrenal weights were also a consistent finding amongthe transgenic animals.

Male transgenic mice bred successfully, while trangenic females showeddecreased fertility. Reduced fertility may be due to elevatedcorticosterone levels or CRF levels in the brain, since both hormoneshave been shown to influence reproductive function [see, for example,Rivier & Vale, in Endocrinology Vol. 114:914-920 (1984); Rivier et al.,in Science Vol. 231:607-609 (1986)].

EXAMPLE 2 Hypothalamic-pituitary-adrenal Axis Hormone Levels in MT-CRFTransgenics

Elevated levels of CRF were not detectable in peripheral blood oftransgenics compared to littermate controls (See Durnam & Palmiter,supra.) To determine whether the increased synthesis of corticosteronein these animals was associated with elevated ACTH values, basal levelsof ACTH and corticosterone were measured in serum obtained fromoffspring derived from a single lineage. In this transgenic line, ACTHvalues were 5-fold elevated compared with those in control nontransgenicanimals. Such increases are well within the range capable of stimulatingincreased synthesis and secretion of corticosterone from the adrenalglands [see Rivier et al., in Endocrinology Vol. 110:272-278 (1982)].Corticosterone levels in transgenic offspring were approximately 10-foldgreater than baseline control (non-transgenic) values. The increases inACTH most likely account for the observed increase in corticosterone.

Because the negative regulation of ACTH expression by glucocorticoidsmay attenuate pituitary responsiveness to chronic CRF stimulation,plasma ACTH levels were determined in the absence of glucocorticoidfeedback. Plasma ACTH levels among adrenalectomized transgenics andcontrols (nontransgenic) were markedly elevated, as expected in asetting devoid of glucocorticoid suppression, but showed no significantdifference between the two groups. This finding may indicate that in theabsence of glucocorticoid, CRF levels in normal and transgenic mice aresufficiently high to drive maximal ACTH production. Interestingly,transgenic mice reverted to a normal phenotype with respect to hair andskin changes within several weeks postadrenalectomy, during which timecirculating corticosterone remained at undetectable levels.

EXAMPLE 3 Tissue Distribution of MT-CRF Transgene Expression

Because the transgenic animals exhibited elevated plasma ACTH levelswithout detectable increases in plasma CRF, the tissue distribution ofCRF gene expression was determined. RNA expression of endogenous CRF insites outside of the central nervous system has been observed at verylow levels in rat testes and adrenal tissue [see Thompson et al., inMol. Endocrinology Vol. 1:363-370 (1987)]. Of the peripheral tissuesexamined by Northern blot analysis, only transgenic testes contained CRFmRNA. Such limited tissue distribution was unexpected, since themajority of MT promoter-regulated transgenes have been reported to betranscribed in liver of kidney. Nevertheless, CRF expression wasundetectable in kidney and liver in four distinct transgenic lines withcushing's syndrome, even in the presence of zinc induction. Mapping ofCRF expression within the testes by in situ hybridization using a ratCRF probe revealed hybridization signal over seminiferous tubules and inan interstitial pattern consistent with Leydig cell expression. Bothgerm cells and Leydig cells have been reported to produce CRF in normalanimals [see Fabbri and Dufau, supra; see also Yoon et al., inEndocrinology Vol. 122:759-761 (1988)]. Further analysis of CRFexpression using reverse transcriptase PCR amplification withtransgene-specific primers revealed expression in testes consistent withthe Northern blot analyses, and in addition, a positive signal was seenin adrenal, heart, and weakly in lung using this more sensitivedetection system. Examination of RNAs isolated from several brain sitesshowed transgene expression in pituitary, hypothalamus, and preopticarea of transgenic brain. To avoid false amplification of the transgenefrom potential genomic DNA contamination in the RNA samples, all sampleswere treated twice with DNAse. In addition, cDNA reactions wereperformed in the presence and absence of reverse transcriptase beforePCR amplification.

EXAMPLE 4 Localization of CRF Expression in Transgenic Mouse Brains

In situ hybridization using a rat CRF probe showed that transgenicanimals have elevated signals for CRF mRNA in nearly all areas ofexpression shared in common with controls. In addition, several regionsnot previously identified as sites of CRF gene or peptide expressioncontained robust mRNA signals, such as the arcuate nucleus of thehypothalamus, the subfornical organ, the lateral habenula, the granulecell layer of the dentate gyrus, the dorsal subiculum, and the deepnuclei of the cerebellum. In contrast to the robust expression of CRFmRNA elsewhere in the central nervous system, the strength of thehybridization signal in the paraventricular nucleus of the hypothalamuswas equivalent, or only marginally elevated, in transgenic animalscompared to that in controls. Because the rat CRF probe does notdistinguish between endogenous mouse and rat does not distinguishbetween endogenous mouse and rat transgene CRF expression, atransgene-specific oligonucleotide probe was used to verify that the CRFtransgene was expressed in regions where high levels or ectopic regionsof CRF expression has been observed. Although the hybridization signalobtained with the oligonucleotide probe is less than that observed usingthe rat CRF riboprobe due to the lowered sensitivity of this method, thetransgene is clearly detectable in transgenic brains and absent incontrol nontransgenic animals. The transgene-specific oligonucleotideprobe revealed a hybridization pattern similar to that detected by therat gene CRF probe, suggesting that the regions of heightened expressionand ectopic sites were due in part to the transgene. Moreover, the factthat the transgene appears to be expressed in the paraventricularnucleus of the hypothalamus, a major site of CRF expression in normalanimals, provides a potential source of CRF overexpression that couldstimulate ACTH production in transgenic animals. In addition, thetransgenic animals displayed CRF mRNA and peptide in the pituitary. Thepositive cells represent approximately 5% of the pituitary cellpopulation. Double immunofluorescence experiments showed that thisexpression does not colocalize with corticotropes; approximately 25% ofthese cells colocalized with FSH-positive cells, indicating partialoverlap with gonadotropes [see Borrelli et al., in Nature Vol.339:538-541 (1989)]. CRF-positive terminals were also observed in theposterior lobe of pituitaries, which is consistent with the CRFexpression seen in magnocellular neurons of the paraventricular andsupraoptic nuclei and provides an additional source of CRF that couldcontribute to the elevated ACTH levels in transgenic mice.

EXAMPLE 5 Evaluation of Animal Behavior

Behavior of the CRF transgenics differed markedly from control animalsin test situations designed to assess behavioral activation and anxietystates. For example, CRF transgenic mice exhibit increased emotionality,consistent with the known anxiogenic effects of centrally administeredCRF. This behavioral anxiety may result from a chronic state of CRFoverproduction, a condition that occurs throughout development and theadult life span of these animals. Behavioral alterations were observedin both the elevated plus-maze and in a novel environment paradigm.These effects appear to be due to central CRF expression sincepre-treatment with the CRF antagonist, α-helical CRF 9-41, reversed thestate of anxiety of transgenic animals.

For example, in the "novel environment" test, locomotor activity variedsignificantly [F(5,100)=25.73, p<0.001]. Analysis of simple effectsindicated that CRF transgenic mice were less active and produced fewercrossovers during the first five minutes of the test [F(1,20)=9.43,p<0.006); F(1,20)=4.35, p<0.05)] bud did not differ significantly fromnontransgenic littermate controls at any subsequent five minute intervalup to 30 minutes (see FIG. 1).

Assessment of locomotor activity in the "novel environment" test showeda clear reduction in locomotion among transgenic animals compared tocontrols. To test whether this behavioral difference could beexaggerated by social defeat stress, animals were tested in the novelenvironment immediately following social defeat by an aggressive malecounterpart mouse. A significant effect was observed during the first 15minutes of testing wherein transgenic animals were markedly hypoactivecompared with unstressed transgenics (see FIG. 2). CRF transgenicsexposed to this social stress were also found to be significantly lessactive than control animals exposed to the same stressor.

The percentage of time spent on exposed vs. enclosed arms [F(1,16=4.56,p<0.05] and the overall activity [F(1,16)=7.53, p<0.02] weresignificantly reduced among CRF transgenic mice relative to controls(see FIG. 3).

The behavior of animals in the novel setting has been shown to besensitive to the effects of acute, central administration of CRF (Suttonet al., supra; Sherman and Kalin, supra). These effects are known tooccur in the absence of HPA activation (Britton et al, supra; Britton etal., supra). The CRF transgenic animals represent an animal modelwherein the transgene is apparently not subject to the regulatorycontrols of the endogenous CRF gene and thus are under continuous CRFstimulation. Elevated CRF expression is accompanied by a markedsuppression in locomotor activity when tested in a novel environment, afinding that parallels the novelty dependent hypoactivity followingcentral CRF infusion. This transient reduction in exploration isprobably not a motor deficit since the locomotor activity of transgenicand littermate control mice did not differ following the initial fiveminute measurement interval. Furthermore, the groups did not differ overthe latter part of the measurement interval in crossover frequency, ameasure of ambulation from one end of the testing environment to theother, suggesting that the activity observed resulted from a normalpattern of exploration. Thus, among the CRF transgenics, continuousexposure to centrally derived CRF results in a behavioral patternsimilar to the anxiogenic-like effects of acute CRF administration.

In order to investigate whether the behavioral effects of novelty couldbe potentiated in the invention animal model by pre-exposure to asupplemental psychological stressor, the effect of a social defeatstressor upon locomotor activity in the novel environment was tested.The locomotor hypoactivity of the CRF transgenics compared with controlanimals subjected to the same compound stressor was severe and morepersistent than that induced by novelty alone. These results indicatethat CRF transgenics display an exaggerated response to stress which isconsistent with an increased state of emotionality.

The behavioral effects of central CRF injection in a variety ofparadigms have been shown to be anxiogenic [Dunn and Berridge, BrainRes. Rev. Vol. 15:71-100 (1990)]. An elevated plus-maze has beenemployed herein as a validated animal model of anxiety. This test, whichis based on the natural aversion of rodents to open spaces, is sensitiveto the effects of both anxiolytic and anxiogenic agents in rats and mice[Pellow et al., supra; Lister, supra; Onaivi et al., J. Pharm. Exp.Ther. Vol. 253:1002-1009 (1990)].and to the stress-protective effects ofa CRF antagonist [Heinrichs et al., Brain Res. Vol. 581:190-197 (1992)].As in the novel environment, clear group differences were observedbetween the CRF transgenics and control animal using this test paradigm.The percentage of time spent on the exposed vs. enclosed arms wassignificantly reduced among the transgenics compared with controlanimals, suggesting that the invention animal model exhibits aspontaneous state of anxiety.

EXAMPLE 6 Effect of ICV Administration of CRF Antagonist on the ElevatedPlus-Maze

To test whether increased emotionality in invention transgenics was due,at least in part, to the expression of CRF in these transgenics, the CRFantagonist, α-helical CRF 9-41, was infused into the lateral ventriclesprior to testing in the elevated plus-maze.

The reduced time spent on the open arms in vehicle-treated CRFtransgenic mice relative to vehicle-treated controls (t(12)=1.8, p<0.05one-tailed) was completely reversed by ICV infusion of the CRFantagonist, α-helical CRF 9-41, at a dose of 5 μg [F(1,12)=17.2,p<0.005] but not 1 μg, five minutes prior to testing on the elevatedplus maze (see FIG. 4A). Among vehicle-treated CRF transgenic mice, theoverall activity score in the Elevated plus maze was suppressed relativeto vehicle-treated controls [t(12)=1.97, p<0.05 one-tailed] whileneither 1 nor 5 μg doses of α-helical CRF 9-41 altered activitysignificantly relative to the respective vehicle-treated groups (seeFIG. 4B).

Administration of a 5 μg dose of the antagonist reversed the significantdecrease in the percentage of time spent on the exposed vs. enclosedarms characteristic of the transgenic mice. These findings support thehypothesis that CRF overproduction in this animal model leads toincreased emotionality.

EXAMPLE 7 Cannulae Placement Histology

Brains from 60% of the cannulated animals were fixed and histologicsections were examined to determine the general accuracy of cannulaplacement. Of those examined, 81% exhibited correct cannula placementwith the needle tract extending through the corpus callosum into theright lateral ventricle. The accuracy of cannula placements among thoseexamined suggests that the present results employing CRF antagonist canbe attributed to infusion into the lateral ventricle.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 3                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other nucleic acid;                                        (A) DESCRIPTION: Oligonucl - #eotide                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - ACAGGAAACT GATGGAGATT ATC           - #                  - #                    23                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other nucleic acid;                                        (A) DESCRIPTION: Oligonucl - #eotide                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - TGGTGGGCAC TGGAGTGGCA ACT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other nucleic acid;                                        (A) DESCRIPTION: Oligonucl - #eotide                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - TTAGGACAAG GCTGGTGGGC ACTGGAGTGG CAACTTCCAG GG    - #                      - #  42                                                                    __________________________________________________________________________

That which is claimed is:
 1. A method of screening for compounds useful in the treatment of Cushing's syndrome, said method comprising administering test compound(s) to a transgenic mouse whose genome comprises a DNA sequence comprising a rat corticotropin releasing factor (CRF) gene operably linked to a mouse metallothionein (MT) promoter, wherein said mouse expresses said gene encoding rat CRF in the paraventricular nucleus of the hypothalamus at a level equivalent, or marginally elevated, as compared to endogenous levels of mouse CRF in the paraventricular nucleus of the hypothalamus of a wild-type mouse, and wherein expression levels of said gene encoding rat CRF are sufficient to effect phenotypic changes consistent with Cushing's disease and anxiety in said mouse, and monitoring for improvement in symptoms characteristic of Cushing's syndrome.
 2. A method of screening for compounds useful in the treatment of anxiety, said method comprising administering test compound(s) to a transgenic mouse whose genome comprises a DNA sequence comprising a rat corticotropin releasing factor (CRF) gene operably linked to a mouse metallothionein (MT) promoter, wherein said mouse expresses said gene encoding rat CRF in the paraventricular nucleus of the hypothalamus at a level equivalent, or marginally elevated, as compared to endogenous levels of mouse CRF in the paraventricular nucleus of the hypothalamus of a wild-type mouse, and wherein expression levels of said gene encoding rat CRF are sufficient to effect phenotypic changes consistent with Cushing's disease and anxiety in said mouse, and monitoring for improvement in symptoms characteristic of anxiety. 